Direct differentiation method for making cardiomyocytes from human embryonic stem cells

ABSTRACT

This invention provides a new procedure for generating cardiomyocyte lineage cells from embryonic stem cells for use in regenerative medicine. Differentiating by way of embryoid body formation or in serum is no longer required. Instead, the stem cells are plated onto a solid substrate, and differentiated in the presence of select factors and morphogens. After enrichment for cells with the appropriate phenotype, the cells are allowed to cluster into Cardiac Bodies™, which are remarkably homogeneous and suitable for the treatment of heart disease.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to provisional patentapplication Ser. No. 60/556,722 filed Mar. 26, 2004; 60/650,194 filedFeb. 3, 2005 and U.S. patent application Ser. No. 11/086,709 filed Mar.21, 2005, now U.S. Pat. No. 7,452,718 B2, Issued Nov. 18, 2008.

Other patent disclosures by Geron Corp. relating to pPS-derivedcardiomyocytes to which this application does not claim priority areU.S. utility application Ser. No. 10/805,099, filed Mar. 19, 2004(099/004p); which is a continuation-in-part of U.S. utility applicationSer. No. 10/193,884 (099/003), filed Jul. 12, 2002, pending; which alongwith International application PCT/US02/22245, filed Jul. 12, 2002 andpublished as WO 03/006950 on Jan. 23, 2003, claims the priority benefitof U.S. provisional applications 60/305,087 (099/001x), filed Jul. 12,2001; and 60/322,695 (099/002x), filed Sep. 10, 2001.

The aforelisted patent disclosures are hereby incorporated herein byreference in its entirety, along with International Patent PublicationsWO 01/51616 (091/200 pct); and WO 03/020920 (091/300 pct), with respectto the culturing and genetic alteration of pPS cells, differentiationinto cardiomyocyte lineage cells, and use of the differentiated cells.

BACKGROUND

A central challenge for research in regenerative medicine is to developcell compositions that can help reconstitute cardiac function. It isestimated that nearly one in five men and women have some form ofcardiovascular disease (National Health and Nutrition Examination SurveyIII, 1988-94, Center of Disease Control and the American HeartAssociation). Widespread conditions include coronary heart disease (5%of the population), congenital cardiovascular defects (0.5%), andcongestive heart failure (3%). The pharmaceutical arts have producedsmall molecule drugs and biological compounds that can help limit thedamage that occurs as a result of heart disease, but there is nothingcommercially available to help regenerate the damaged tissue.

With the objective of developing a cell population capable of cardiacregeneration, research has been conducted on several different fronts.Clinical trials are underway at several centers to test the use ofautologous bone marrow derived cells for therapy after myocardialinfarction (Perin et al., Circulation 107:2294, 2003; Strauer et al.,Circulation 106:1913, 2002; Zeiher et al., Circulation 106:3009, 2002;Tse et al., Lancet 361:47, 2003; Stamm et al., Lancet 3661:45, 2003). Ithas been hypothesized that the cells may have a cleansing function toimprove blood perfusion of the heart tissue. Clinical trials are alsounderway to test the use of autologous skeletal muscle myoblasts forheart therapy (Menasche et al., J. Am. Coll. Cardiol, 41:1078, 2003;Pagani et al., J. Am. Coll. Cardiol. 41:879, 2003; Hagege et al., Lancet361:491, 2003). However, it is unclear if the contraction of striatalmuscle cells can coordinate adequately with cardiac rhythm.

A more direct approach would be to use cells that are already committedto be functional cardiomyocytes. Syngeneic neonatal or postnatal cardiaccells have been used in animal models to repair damage resulting frompermanent coronary occlusion (Reffelmann et al., J. Mol. Cell Cardiol.35:607, 2003; Yao et al., J. Molec. Cell. Cardiol. 35:607, 2003.Accordingly, if such cells were available for human therapy, they couldbe very effective for the treatment of ischemic heart disease.

International patent publication WO 99/49015 (Zymogenetics) proposes theisolation of a nonadherent pluripotent cardiac-derived human stem cell.Heart cells are suspended, centrifuged on a density gradient, cultured,and tested for cardiac-specific markers. Upon proliferation anddifferentiation, the claimed cell line produces fibroblasts, musclecells, cardiomyocytes, keratinocytes, osteoblasts, or chondrocytes.However, it is unclear whether any of the cell preparations exemplifiedin these publications can be produced in sufficient quantities for massmarketing as a therapeutic composition for regenerating cardiacfunction.

A potential source of regenerative cells for treating cardiac disease ispluripotent stem cells of various kinds, especially embryonic stemcells. Several laboratories have reported results using mouse ES cells(Wobus et al., J. Mol. Cell Cardiol. 29:1525, 1997; Kolossov et al., J.Cell Biol. 143:2045, 1998; Narita et al., Development 122:3755, 1996; L.Field, U.S. Pat. No. 6,015,671; Klug et al., J. Clin. Invest. 98:216,1996; Doevendans et al., J. Mol Cell Cardiol. 32:839, 2000; Muller etal., FASEB J. 14:2540, 2000; Gryschenko et al., Pflugers Arch. 439:798,2000).

Thomson et al. (Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the firstto successfully culture embryonic stem cells from primates, using rhesusmonkeys and marmosets as a model. They subsequently derived humanembryonic stem (hES) cell lines from human blastocysts (Science 282:114,1998). Human embryonic stem cells can proliferate in vitro withoutdifferentiating; they retain a normal karyotype, and the capacity todifferentiate to produce a variety of adult cell types.

However, a number of obstacles have stood in the way of developing aparadigm for obtaining substantially enriched populations ofcardiomyocyte lineage cells from primate pluripotent stem (pPS) cells.Some ensue from the relative fragility of pluripotent cells of primateorigin, the difficulty in culturing them, and their exquisitesensitivity and dependence on various factors present in the cultureenvironment. Other obstacles ensue from the understanding that cardiacprogenitor cells require visceral embryonic endoderm and primitivestreak for terminal differentiation (Arai et al., Dev. Dynamics 210:344,1997). In order to differentiate pPS cells into cardiac progenitor cellsin vitro, it is necessary to mimic or substitute for all the events thatoccur in the natural ontogeny of such cells in the developing fetus.

Small patches of beating cells can be generated from hES cells by ageneralized differentiation protocol, and it has recently been proposedthat these cells be used for determining the effect of small moleculedrugs of cardiomyocyte transmembrane potentials (WO 04/011603; Thomson,Kamp et al.). It has been proposed that differentiated cell populationscontaining a few cardiac cells can be generated simply by culturing in amedium supplemented with serum, and then somehow sorting out the beatingcells (WO 04/081205; ES Cell International). It is unclear how cellpopulations having a low frequency of cardiomyocyte lineage cells can beused to generate preparations sufficiently pure for therapeutic use in acommercially viable manner.

Geron Corporation has developed novel tissue culture environments thatallow for continuous proliferation of human pluripotent stem cells in anenvironment essentially free of feeder cells (see U.S. Pat. No.6,800,480; Australian patent AU 751321, and International PatentPublication WO 03/020920). Feeder-free pPS cell cultures can be used tomake differentiated cell populations free of xenogeneic contaminants,such as hepatocytes (U.S. Pat. No. 6,458,589), neural cells (U.S. Pat.No. 6,833,269), and cardiomyocytes (WO 01/88104).

Commercialization of these technologies for use in regenerative medicinewill benefit from further improvement in the expansion anddifferentiation protocols to improve cell homogeneity and yield.

SUMMARY

This invention provides a system for differentiating pluripotent stemcells of human origin into differentiated cell populations that arehighly enriched for cardiomyocyte lineage cells—either end-stagecardiomyocytes, or cardiomyocyte precursors capable of proliferation invitro and capable of further differentiation in vitro or in vivo intotherapeutically useful phenotypes.

The new differentiation method of this invention for obtaining enrichedpopulations of cardiomyocyte lineage cells from primate pluripotent stem(pPS) cells has several aspects. One is to initiate the process by adirect differentiation protocol. Undifferentiated pPS cells are platedwithout forming embryoid bodies directly onto a solid surface comprisinga substrate to which cardiomyocyte lineage cells adhere (such as gelatinor fibronectin). The plated cells are cultured for a time with aspecific factor combination that directs the cells into thecardiomyocyte pathway with high fidelity. Exemplary are activins andbone morphogenic proteins, particularly BMP-4, typically used in theabsence of retinoic acid or serum. The factors can then be withdrawn andthe culture continued. The presence of the substrate and the factors canrender unnecessary the use of serum containing components or feedercells (i.e., any cells having a different phenotype and genome that mayact to control differentiation). The harvested cell population issubstantially enriched for cardiomyocyte lineage cells and cardiacprecursors, and can be treated further to increase the proportion ofcells with a cardiac phenotype (e.g., expression of α-cardiac myosinheavy chain). Exemplary techniques include separation on a densitygradient such as Percoll™, or immunological separation using cellsurface markers listed in this disclosure.

The proportion of cardiac cells is further increased by the formation ofCardiac Bodies™. These are clusters of cardiac cells in suspension, manyof which undergo spontaneous contraction. In an exemplary method, pPSderived cell populations are first prepared comprising a substantialproportion of cells expressing characteristics of the cardiomyocytelineage. The cells are suspended in culture medium, and single cells areremoved, leaving cells that are present as clusters. The clustered cellsare then resuspended and recultured in fresh medium for a suitableperiod. The cells can be taken through multiple cycles of separating,resuspending, and reculturing, until a composition is obtained in whichup to 80 or 100% of the cell clusters undergo spontaneous contraction.The invention embodies methods of manufacturing Cardiac Bodies™ from pPScells and mixed populations of cardiomyocyte lineage cells, andcompositions of the Cardiac Bodies™ themselves, optionally in the formof a cultured cell composition, or a composition suitable foradministration to a mammalian subject.

One use of the invention is the screening of a compound for an effect oncardiomyocytes. This involves combining the compound with a cellpopulation of the invention, and then determining any modulatory effectresulting from the compound. This may include examination of the cellsfor toxicity, metabolic change, or an effect on contractile activity.

Another use of the invention is the formulation of cardiomyocyte lineagecells as a medicament or in a delivery device intended for treatment ofa human or animal body. This enables the clinician to administer thecells in or around the damaged heart tissue either from the vasculatureor directly into the muscle wall, thereby allowing the heart cells toengraft, limit the damage, and participate in regrowth of the heartmusculature and restoration of cardiac function.

These and other embodiments of the invention will be apparent from thedescription that follows.

DRAWINGS

FIG. 1 shows single cells and cell clusters separated and stained fortropomyosin, titin, myosin heavy chain (MHC), α-actinin, desmin, cardiactroponin I (cTnI), and cardiac troponin T (cTnT). Single cells andclusters stained positive for all these markers. The striationscharacteristic of the sarcomeric structures can be seen, a feature thatis consistent with the ability of the cells to exhibit contractileactivity.

FIG. 2 shows the effect of pharmacological agents on contractileactivity of the hES derived cardiomyocytes. The L-type calcium channelinhibitor diltiazem inhibited contractile activity in a dose-dependentfashion. The adrenoceptor agonists isoprenaline, phenylephrine, andclenbuterol had a chronotropic effect.

FIG. 3 illustrates the evaluation of potential cardiotropic factors fortheir ability to enhance the proportion of cardiomyocyte lineage cellsin the population. Activins and certain growth factors were introducedduring embryold body formation (Group I); other growth factors (GroupII) and 5-aza-deoxy-cytidine were introduced after plating onto gelatin;and additional factors (Group II) were added later duringdifferentiation. The combinations were tested at three concentrationlevels. Most effective were low concentrations of growth factors incombination with 5-aza-deoxy-cytidine.

FIGS. 4(A) and 4(B) show further refinement of the protocol by adjustingeach group of factors independently. The A-MHC marker characteristic ofcardiomyocytes was most abundantly produced when the factors in Groups Iand II were used at low levels and followed by 5-aza-deoxy-cytidine.Group III factors used later during differentiation actually inhibitedcardiomyocyte formation. Expression of the earlycardiomyocyte-associated gene GATA-4 was also improved under theseconditions. The effect on α-MHC and GATA-4 was selective, in comparisonwith the endoderm-associated gene HNF3b, which increased using anygrowth factor combination.

FIG. 5(A) shows the expression of cardiomyocyte phenotype in cellsproduced by direct differentiation of hES cells. Undifferentiated hEScells were grown to confluence on a substrate of gelatin coated withFBS, induced to differentiate using Activin A and BMP-4 in a serum-freemedium, and then cultured in the absence of the differentiation factorsfor 14 days. Cell populations were obtained that expressed substantiallyhigher levels of myosin heavy chain, compared with cells generated fromembryoid bodies in serum-containing medium in the usual fashion.Numerous spontaneously beating areas were evident 7 days after theremoval of Activin A and BMP-4. FIG. 5(B) shows staining of the cellsfor the transcription factor Nkx2.5 and α-sarcomeric actinin,characteristic of cardiomyocyte lineage cells.

FIG. 6 is taken from an experiment in which the hES cells were expandedin defined medium before being used to make the cardiomyocytes by directdifferentiation.

FIG. 7 shows the expression of cTnI measured in Cardiac Bodies™ formedfrom each of the four Percoll™ fractions. Undifferentiated hES cells areused as a negative control. Culturing the Fraction IV cells as CardiacBodies™ enriched for αMHC or cTnI expression by 100- to 500-fold.

FIG. 8(A) shows a field of Cardiac Bodies™ made from Fraction IV cells(bar≡300 μm). The clusters marked by the arrows were undergoingspontaneous contractions. FIG. 8(B) shows the proportion of clustersthat were beating when Cardiac Bodies™ were made from each of thePercoll™ fractions, following 12 or 20 days of differentiation. Thecombination of a 20 day differentiation period, separation of thehighest density fraction, and subsequent culturing of the CardiacBodies™ for 7 days produced the highest proportion of clustersundergoing spontaneous contraction.

FIG. 9 shows that cardiomyocyte lineage cells of this invention canintegrate and persist in the myocardium when administered in vivo. TheH&E staining (Top Panel) is compared with staining with a human-specificpancentromeric probe (Middle Panel), and an antibody specific for humanβ-myosin heavy chain (Lower Panel), showing the presence of hES derivedcardiomyocytes in the engrafted area.

DETAILED DESCRIPTION

This invention provides a improved protocols, techniques, and reagentsfor preparing and characterizing cardiomyocytes and their precursorsfrom primate pluripotent stem cells.

Previous patent applications and publications in this series provideprocedures for differentiating primate pluripotent stem (pPS) cells intocardiomyocyte lineage cells (WO 03/006950; Xu et al., Circ. Res. 91:501,2002). pPS derived cell populations were obtained that contain cellswere positive for markers such as myosin heavy chain (MHC) and cardiactroponin I (cTnI), and that undergo spontaneous periodic contraction intissue culture.

The protocols provided in the following description incorporate severalimportant advances that improve the cardiomyocyte production process.First of all, it has been discovered that cardiomyocytes can be madefrom completely undifferentiated pPS cells directly on a solid surfaceor matrix. This avoids the need to initiate the differentiation processby making embryoid bodies, improving the uniformity of the cellsobtained. Direct differentiation involves culturing with certaincardiotropic factors and morphogens that direct the cells into thecardiomyocyte lineage (Example 5)—an event that is normally controlledby cross-cellular signaling in the embryo or embryoid body. It has alsobeen discovered that the process can be further enhanced by withdrawingthe factors and continuing the culture for a time, which not onlyexpands the cell population, but surprisingly improves the yield ofearly-stage cardiomyocyte lineage cells. Without implying any limitationon the nature or use of the cell populations, it is proposed that thepresence of early stage cells helps enhance the ability of the cells toestablish, adapt, or proliferate in vivo, which in turn enhances theirability to regenerate cardiac tissue in a beneficial way.

Another important process development comes from the observation thatcardiomyocyte lineage cells generated from pPS cells can be made tocluster in culture. These clusters are referred to in this disclosure asCardiac Bodies™. It has been discovered that allowing cardiac bodies toform, dispersing them, and then repeating the process in multiple cyclesconsiderably enhances the proportion of cells having a desirablephenotype (Examples 6 and 7). This process is particularly well adaptedto commercial scale-up. In addition, the clusters may be more stable instorage, and provide a more effective source of cardiomyocytes for usein regenerative medicine.

Further advances in the making of cardiomyocyte cell populations arealso described below. The remarkable uniformity and functionalproperties of the cells produced according to this disclosure make themvaluable for studying cardiac tissue in vitro, and for developing newtherapeutic modalities for regeneration of cardiac tissue in thetreatment of heart disease.

DEFINITIONS

The techniques and compositions of this invention relate to pPS-derivedcardiomyocytes and their precursors. Phenotypic characteristics ofcardiomyocytes are provided in a later section of this disclosure. Thereare no particular characteristics that are definitive for cardiomyocyteprecursors, but it is recognized that in the normal course of ontogeny,undifferentiated pPS cells first differentiate into mesodermal cells,and then through various precursor stages to a functional (end-stage)cardiomyocyte.

Accordingly, for the purposes of this disclosure, a “cardiomyocyteprecursor” is defined as a cell that is capable (withoutdedifferentiation or reprogramming) of giving rise to progeny thatinclude mature (end-stage) cardiomyocytes. Cardiomyocyte precursor cellscan often be identified using one or more markers selected from GATA-4,Nkx2.5, and the MEF-2 family of transcription factors.

The term “cardiomyocyte lineage cells” refers generally to bothcardiomyocyte precursor cells and mature cardiomyocytes. Reference tocardiomyocyte lineage cells, precursors, or cardiomyocytes in thisdisclosure an be taken to apply equally to cells at any stage ofcardiomyocyte ontogeny without restriction, as defined above, unlessotherwise specified. As described below, cardiomyocyte linage cells mayhave one or more markers (sometimes at least 3 or 5 markers) from thefollowing list: cardiac troponin I (cTnI), cardiac troponin T (cTnT),sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin,β1-adrenoceptor (β1-AR), ANF, the MEF-2 family of transcription factors,creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor(ANF).

Certain cells of this invention demonstrate spontaneous periodiccontractile activity. This means that when they are cultured in asuitable tissue culture environment with an appropriate Ca⁺⁺concentration and electrolyte balance, the cells can be observed tocontract in a periodic fashion across one axis of the cell, and thenrelease from contraction, without having to add any additionalcomponents to the culture medium.

The name Cardiac Body™ (used in the singular or plural) has been createdby Geron Corporation as a term or brand for a cardiomyocyte cluster—morespecifically, a cluster of pPS derived cells in suspension, bearing twoor more characteristics of human cardiomyocyte lineage cells. Asubstantial proportion of cells in the cluster express cTnI, cTnT, ANF,or MHC from an endogenous gene, and the cluster usually undergoesspontaneous contraction in the presence of Ca⁺⁺ and appropriateelectrolytes. The cardiomyocyte cluster may be present in a cellculture, in a pharmaceutical preparation, or any other usefulcomposition. This disclosure allows the user to prepare suspensions ofCardiac Bodies™ in which well over 50% undergo spontaneous contraction

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells derived from any kind of embryonic tissue (fetal or pre-fetaltissue), and have the characteristic of being capable under appropriateconditions of producing progeny of different cell types that arederivatives of all of the 3 germinal layers (endoderm, mesoderm, andectoderm), according to a standard art-accepted test, such as theability to form a teratoma in 8-12 week old SCID mice, or the ability toform identifiable cells of all three germ layers in tissue culture.

Included in the definition of pPS cells are embryonic cells of varioustypes, exemplified by human embryonic stem (hES) cells, described byThomson et al. (Science 282:1145, 1998); embryonic stem cells from otherprimates, such as Rhesus stem cells (Thomson et al., Proc. Natl. Acad.Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol.Reprod. 55:254, 1996) and human embryonic germ (hEG) cells (Shamblott etal., Proc. Natl. Acad. Sci. USA 95:13726, 1998). These cell types may beprovided in the form of an established cell line, or they may beobtained directly from primary embryonic tissue and used immediately fordifferentiation. Other types of pluripotent cells are also included inthe term. Any cells of primate origin that are capable of producingprogeny that are derivatives of all three germinal layers are included,regardless of whether they were derived from embryonic tissue, fetaltissue, or other sources. The pPS cells are not derived from a malignantsource. It is desirable (but not always necessary) that the cells bekaryotypically normal.

pPS cell cultures are described as “undifferentiated” when a substantialproportion of stem cells and their derivatives in the population displaymorphological characteristics of undifferentiated cells. It isunderstood that colonies of undifferentiated cells within the populationwill often be surrounded by neighboring cells that are partlydifferentiated.

The term “embryoid bodies” refers to heterogeneous clusters comprisingdifferentiated and partly differentiated cells that appear when pPScells are allowed to differentiate in a non-specific fashion insuspension cultures or aggregates.

“Direct differentiation” refers to a process for differentiating pPScells into progeny that are enriched for cells of a particular tissuetype without forming embryoid bodies as an intermediate. This may bedone when the cells are plated on a solid substrate, although plating isnot necessarily required if not explicitly specified. Directdifferentiation is effected by culturing in a growth environment ofmedia components, soluble factors, insoluble components in suspension oron the vessel wall, and other ingredients that accomplish the objectiveof directing the cells towards the desired tissue type.

“Feeder cells” are cells of a different tissue type and typically adifferent genome that may act to promote proliferation and/or controldifferentiation of cells they are cocultured with. Undifferentiated pPScells can be cocultured with feeders that help maintain theundifferentiated state, while cells being differentiated can becocultured with feeders that direct differentiation towards a particulartissue type (e.g., cardiomyocytes). The techniques described in thisdisclosure can be employed in the absence of feeder cells of eitherkind.

A “growth environment” is an environment in which cells of interest willproliferate, differentiate, or mature in vitro. Features of theenvironment include the medium in which the cells are cultured, anygrowth factors or differentiation-inducing factors that may be present,and a supporting structure (such as a substrate on a solid surface) ifpresent.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, embryology, andcardiophysiology.

With respect to tissue culture and embryonic stem cells, the reader maywish to refer to Teratocarcinomas and embryonic stem cells: A practicalapproach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide toTechniques in Mouse Development (P. M. Wasserman et al. eds., AcademicPress 1993); Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles,Meth. Enzymol. 225:900, 1993); Properties and uses of Embryonic StemCells: Prospects for Application to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998). With respect tothe culture of heart cells, standard references include The Heart Cellin Culture (A. Pinson ed., CRC Press 1987), Isolated AdultCardiomyocytes (Vols. I & II, Piper & Isenberg eds., CRC Press 1989),Heart Development (Harvey & Rosenthal, Academic Press 1998), I Left myHeart in San Francisco (T. Bennet, Sony Records 1990); and Gone with theWnt (M. Mitchell, Scribner 1996). General methods in molecular andcellular biochemistry can be found in such standard textbooks as ShortProtocols in Molecular Biology, 4th Ed.; Immunology Methods Manual (I.Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture:Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley &Sons 1998).

Sources of Stem Cells

This invention can be practiced with pluripotent stem cells of varioustypes, particularly stem cells derived from embryonic tissue and havethe characteristic of being capable of producing progeny of all of thethree germinal layers, as described above.

Exemplary are embryonic stem cells and embryonic germ cells used asexisting cell lines or established from primary embryonic tissue of aprimate species, including humans. This invention can also be practicedusing pluripotent cells obtained from primary embryonic tissue, withoutfirst establishing an undifferentiated cell line.

Embryonic Stem Cells

Embryonic stem cells can be isolated from blastocysts of primate species(U.S. Pat. No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA92:7844, 1995). Human embryonic stem (hES) cells can be prepared fromhuman blastocyst cells using the techniques described by Thomson et al.(U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol.38:133 ff., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000.Equivalent cell types to hES cells include their pluripotentderivatives, such as primitive ectoderm-like (EPL) cells, outlined in WO01/51610 (Bresagen).

hES cells can be obtained from human preimplantation embryos (Thomson etal., Science 282:1145, 1998). Alternatively, in vitro fertilized (IVF)embryos can be used, or one-cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos arecultured to the blastocyst stage, the zona pellucida is removed, and theinner cell masses are isolated (for example, by immunosurgery usingrabbit anti-human spleen cell antiserum). The intact inner cell mass isplated on mEF feeder cells (U.S. Pat. No. 5,843,780), human feeder cells(US 2002/0072117 A1), or in a suitable feeder free environment thatsupports undifferentiated hES cell growth (US-2002-0081724-A1; WO03/020920). Growing colonies having undifferentiated morphology aredissociated into clumps, and replated.

Propagation of pPS Cells in an Undifferentiated State

pPS cells can be propagated continuously in culture, using cultureconditions that promote proliferation while inhibiting differentiation.Exemplary serum-containing ES medium is made with 80% DMEM (such asKnock-Out DMEM, Gibco), 20% of either defined fetal bovine serum (FBS,Hyclone) or serum replacement (US 2002/0076747 A1, Life TechnologiesInc.), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol.

Traditionally, ES cells are cultured on a layer of feeder cells,typically fibroblasts derived from embryonic or fetal tissue (Thomson etal., Science 282:1145, 1998). Scientists at Geron have discovered thatpPS cells can be maintained in an undifferentiated state even withoutfeeder cells. The environment for feeder-free cultures includes asuitable culture substrate, particularly an extracellular matrix such aslaminin or Matrigel® (basement membrane produced byEngelbreth-Holm-Swarm tumor cells and containing extracellular matrixcomponents such as laminin). The pPS cells are plated at >15,000 cellscm⁻² (optimally 90,000 cm⁻² to 170,000 cm⁻²). Typically, enzymaticdigestion is halted before cells become completely dispersed (say, ˜5min with collagenase IV). Clumps of ˜10 to 2,000 cells are then plateddirectly onto the substrate without further dispersal. Alternatively,the cells can be harvested without enzymes before the plate reachesconfluence by incubating ˜5 min in a solution of 0.5 mM EDTA in PBS.After washing from the culture vessel, the cells are plated into a newculture without further dispersal. In a further illustration, confluenthES cells cultured in the absence of feeders are removed from the platesby incubating with a solution of 0.05% (wt/vol) trypsin (Gibco) and0.053 mM EDTA for 5-15 min at 37° C. The remaining cells in the plateare removed and the cells are triturated into a suspension comprisingsingle cells and small clusters, and then plated at densities of50,000-200,000 cells cm⁻² to promote survival and limit differentiation.

Feeder-free cultures are supported by a nutrient medium containingfactors that promote proliferation of the cells without differentiation(U.S. Pat. No. 6,800,480). Such factors may be introduced into themedium by culturing the medium with cells secreting such factors, suchas irradiated (˜4,000 rad) primary mouse embryonic fibroblasts,telomerized mouse fibroblasts, or fibroblast-like cells derived from pPScells (U.S. Pat. No. 6,642,048). Medium can be conditioned by platingthe feeders in a serum free medium such as KO DMEM supplemented with 20%serum replacement and 4 ng/mL bFGF. Medium that has been conditioned for1-2 days is supplemented with further bFGF, and used to support pPS cellculture for 1-2 days (WO 01/51616; Xu et al., Nat. Biotechnol. 19:971,2001).

Alternatively, fresh or non-conditioned medium can be used, which hasbeen supplemented with added factors (like a fibroblast growth factor orforskolin) that promote proliferation of the cells in anundifferentiated form. Exemplary is a base medium like X-VIVO™ 10(Biowhittaker) or QBSF™-60 (Quality Biological Inc.), supplemented withbFGF at 40-80 ng/mL, and optionally containing stem cell factor (15ng/mL), or Flt3 ligand (75 ng/mL). These medium formulations have theadvantage of supporting cell growth at 2-3 times the rate in othersystems (WO 03/020920).

Under the microscope, ES cells appear with high nuclear/cytoplasmicratios, prominent nucleoli, and compact colony formation with poorlydiscernable cell junctions. Primate ES cells typically express thestage-specific embryonic antigens (SSEA) 3 and 4, and markers detectableusing antibodies designated Tra-1-60 and Tra-1-81. Undifferentiated hEScells also typically express the transcription factor Oct-3/4, Cripto,gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein(PODXL), and human telomerase reverse transcriptase (hTERT) (US2003/0224411 A1), as detected by RT-PCR.

Procedures for Preparing Cardiomyocytes

Cardiomyocyte lineage cells can be obtained from undifferentiated stemcells by culturing or differentiating in a special growth environmentthat enriches for cells with the desired phenotype (either by outgrowthof the desired cells, or by inhibition or killing of other cell types).

Differentiation can be initiated by forming embryoid bodies oraggregates: for example, by overgrowth of a donor pPS cell culture, orby culturing pPS cells in suspension in culture vessels having asubstrate with low adhesion properties which allows EB formation. pPScells are harvested by brief collagenase digestion, dissociated intoclusters, and plated in non-adherent cell culture plates (WO 01/51616;U.S. Pat. No. 6,602,711). Optionally, the EBs can be producedencapsulated in alginate or other suitable nutrient-permeable matrix,which may help improve the uniformity of EB diameter and consistency ofthe cells produced (WO 03/004626, Zandstra et al.). Whether or not theprocess involves EB formation, using a medium that contains serum orserum equivalent promotes foci of contracting cells of the cardiomyocytelineage: for example, ˜20% fetal bovine serum, or a serum supplementsuch as B27 or N2 in a suitable growth medium such as RPMI.

To promote the cardiomyocyte phenotype, the cells can be cultured withfactors and factor combinations that enhance proliferation or survivalof cardiomyocyte type cells, or inhibit the growth of other cell types.The effect may be due to a direct effect on the cell itself, or due toan effect on another cell type, which in turn enhances cardiomyocyteformation. For example, factors that induce the formation of hypoblastor epiblast equivalent cells, or cause these cells to produce their owncardiac promoting elements, all come within the rubric of cardiotropicfactors.

Factors thought to induce differentiation of pPS cells into cells of themesoderm layer, or facilitate further differentiation into cardiomyocytelineage cells include the following:

-   -   Transforming Growth Factor beta related ligands (exemplified by        TGF-β1, TGF-β2, TGF-β3 and other members of the TGF-β        superfamily illustrated below). Ligands bind a TGF-β receptor        activate Type I and Type II serine kinases and cause        phosphorylation of the Smad effector.    -   Morphogens like Activin A and Activin B (members of the TGF-β        superfamily)    -   Insulin-like growth factors (such as IGF I and IGF II)    -   Bone morphogenic proteins (members of the TGF-β superfamily,        exemplified by BMP-2 and BMP-4)    -   Fibroblast growth factors (exemplified by bFGF, FGF-4, and        FGF-8), other ligands that activate cytosolic kinase raf-1 and        mitogen-activated proteins kinase (MAPK), and other mitogens        such as epidermal growth factor (EGF)    -   Nucleotide analogs that affect DNA methylation and altering        expression of cardiomyocyte-related genes (e.g.,        5-aza-deoxy-cytidine)    -   The pituitary hormone oxytocin, or nitric oxide (NO)    -   Specific antibodies or synthetic compounds with agonist activity        for the same receptors

Particularly effective combinations of cardiotropic agents include useof a morphogen like Activin A and a plurality of growth factors, such asthose included in the TGF-β and IGF families during the early commitmentstage, optionally supplemented with additional cardiotropins such as oneor more fibroblast growth factors, bone morphogenic proteins, andplatelet-derived growth factors.

During the elaboration of this invention, it was found that omittingfactors such as insulin-like growth factor II (IGF II) and relatedmolecules from the final stages of in vitro differentiation actuallyincrease the levels of cardiac gene expression. In unrelated studies,IGF II has been found to decrease the levels of GSK3β in fibroblasts(Scalia et al., J. Cell. Biochem. 82:610, 2001). IGF II may thereforepotentiate the effects of Wnt proteins present in the culture medium orsecreted by the cells. Wnt proteins normally stabilize and cause nucleartranslocation of a cytoplasmic molecule, β catenin, which comprises aportion of the transcription factor TCF. This changes transcriptionalactivity of multiple genes. In the absence of Wnt, β catenin isphosphorylated by the kinase GSK3β, which both destabilizes β cateninand keeps it in the cytoplasm.

Since Wnt activators like IGF II apparently limit cardiomyocytedifferentiation, this invention includes culturing with Wnt antagoniststo increase the extent or proportion of cardiomyocyte differentiation ofpPS cells. Wnt signaling can be inhibited by injection of synthetic mRNAencoding either DKK-1 or Crescent (secreted proteins that bind andinactivate Wnts) (Schneider et al., Genes Dev. 15:304, 2001), or byinfection with a retrovirus encoding DKK-1 (Marvin et al., Genes Dev.15:316, 2001). Alternatively, the Wnt pathway can be inhibited byincreasing the activity of the kinase GSK3β, for example, by culturingthe cells with factors such as IL-6 or glucocorticoids.

Evaluation of potential cardiotropic agents is illustrated in Example 3.Of course, unless explicitly required, it is not necessary to understandthe mode of action of a cardiotropic factor in order to employ it in adifferentiation paradigm according to this invention. The combinationsand amounts of such compounds that are effective for enrichingcardiomyocyte production can be determined empirically by culturingundifferentiated or early differentiated hES cells or their progeny in aculture environment incorporating such factors, and then determiningwhether the compound has increased the number of cardiomyocyte lineagecells in the population according to the phenotypic markers listedbelow.

Direct Differentiation

As already described, differentiation paradigms for pPS cellstraditionally involve forming embryoid bodies, which allows cross-talkbetween different cell types, thought to promote tissue formation in amanner reminiscent of an embryo. However, it is often advantageous toeliminate the need to form embryoid bodies, allowing the differentiationprocess to be more controlled, and the resulting cell population tend tobe more uniform (WO 01/51616; US 2002/0151053 A1). This disclosureprovides new methods for direct differentiation of hES cells intocardiomyocytes, without forming embryoid bodies and without using serumor serum supplements.

An illustration of the direct differentiation technique is provided inExample 5. First, the pPS cells are harvested from the culture in whichthey are expanded (optimally feeder-free), and plated onto a substrateor matrix that is adherent for undifferentiated hES cells, and iscompatible with cardiomyocyte differentiation. Exemplary are 0.5%gelatin, 20 μg/mL fibronectin, or Matrigel®. The substrate or matrix canbe coated onto the surface of the culture vessel; or in some instancescan be part of a particulate or meshwork support present throughout theculture environment. When gelatin is used, adherence of the cells can bepromoted by preincubating the matrix with serum, and then washing awaythe serum before plating the cells. If desired, the pPS cells can beestablished onto the substrate before initiating differentiation—forexample, by continuing to culture for a suitable time (say, 4 to 8 days)with a similar medium to what is used to expand the pPS cells in theundifferentiated form. This will typically bring the pPS cells toconfluence as a monolayer in the new culture environment.

The differentiation process is initiated by culturing the plated cellsin a medium that contains factors referred to elsewhere in thisdisclosure that promote cardiomyocyte differentiation. Exemplary areactivins and/or bone morphogenic proteins. A combination of TGF-βsuperfamily proteins Activin A and BMP-4 is particularly effective(Example 5). In some circumstances, other morphogens like BMP-2 maysubstitute for BMP-4. The medium used at this stage or for later culturesteps may contain adjuncts selected from the list provided earlier.Exemplary are insulin like growth factors, particularly IGF I, and/or atumor necrosis factor or other inflammatory cytokine, particularlyTNF-α. Culturing with the differentiation factors can take anywhere froma few days to several weeks or more to direct the cells into thecardiomyocyte lineage, with 4-7 days being typical.

One of the advantages of this technique is that a serum or serumsubstitute is not needed to initiate or support the cardiomyocytedifferentiation process, as is typical of other methods. Instead, themedium can be formulated so that it contains an artificial nutritionalsupplement that supports differentiated cells like cardiomyocytes orneurons. Exemplary are B27 supplement, N2 supplement, and G5 supplement(Life Technologies/Gibco). Such supplements often comprise nutrients andcofactors like human insulin (500 μg/L), human transferrin (5-10 mg/mL),and selenium (0.5 μg/mL), and may also contain putrescine (1.5 mg/L),biotin (1 μg/L), hydrocortisone (0.4 μg/L), or progesterone (0.6 μg/L),and/or low levels of mitogens like EGF or FGF (1 μg/L). For purposes ofcommercial scale production and human therapy, elimination of componentsderived from non-human animals is particularly advantageous.

It has also been found that the proportion of cardiomyocyte lineagecells suitable for regenerative medicine can often be enhanced bywithdrawing the TGF-β superfamily morphogens, and then continuing theculture for a few days or as many as 1 or 2 weeks or more in a similarsupplemented medium. In this step, the medium sometimes can containgrowth factors such as IGF, but BMP-2, other BMPs, or other morphogensmay delay emergence of the cardiomyocyte phenotype and reduce yield.

Cardiomyocyte lineage cells (identified by marker expression orcontraction activity) are ultimately harvested from the culture. Theharvested population may contain over 5% or 10% Nkx2.5 positive cells.

Populations of differentiated cells can then be further processed toenrich cells with desirable characteristics, such as by mechanicalseparation or sorting for surface markers. For example, the percentageof contracting cells can be enriched up to ˜20-fold by densityseparation. Isolation of enriched cardiomyocyte populations by isopycniccentrifugation is illustrated in Examples 1 and 4. Populations can beobtained that comprise at least ˜5%, ˜20%, ˜60%, and potentially over˜90% cells of the cardiomyocyte lineage, identified by expression of MHCor other tissue specific marker. Many of the research and therapeuticapplications referred to in this disclosure benefit from enrichment ofthe proportion of cardiomyocytes, but complete homogeneity is often notrequired.

Formation of Cardiac Bodies™

It also has been discovered that preparations of pPS derivedcardiomyocytes can be further expanded or enriched by allowing them togrow in clusters that are referred to as Cardiac Bodies™.

First, a cell population is generated that contains cells with phenotypecharacteristics of cardiomyocyte lineage cells, and optionally enrichedby density separation or other technique. The cells are then allowed toform clusters, and single cells in the suspension are removed. This canbe accomplished by letting the clusters settle, and pipetting out thesupernatant containing single cells. Before proceeding, it is sometimesbeneficial to break apart the clusters (for example, by brieftrypsinization and/or mechanical dispersion). The cells are thencultured in suspension in low adhesion plates in fresh culture medium(exemplified by medium containing fetal bovine serum, serum substitute,or CCT as described earlier), and allowed to reaggregate into“secondary” Cardiac Bodies™. Culturing then continues with periodicrefeeding, as necessary, with cardiomyocyte lineage cells remaining asclusters of 10 to 5000 cells (typically 50 to 1000 cells) in size.

After a suitable period (typically 1 to 7 days), the cultured cells canbe harvested for characterization, or used in drug screening orpharmaceutical manufacture. The purification effect may improve if thecells are taken through further cycles of removing single cells andreculturing the clusters, over a period of 8 days or more. Each cyclecan optionally incorporate a step in which the clusters of cells aredispersed into single cells, or smaller cell clusters, to allow forfurther expansion. Larger clusters may form, either by aggregation ofthe suspended cells, or by proliferation within the cluster, or both. Itis a hypothesis of this invention that cardiomyocyte lineage cells havea tendency to form such clusters under appropriate conditions, and thatthe removal of single cells helps eliminate other cell types andincrease homogeneity.

Examples 6 and 7 illustrate the process. Mixed populations of cellscontaining cardiomyocytes were put in fresh medium, and the clusterswere harvested by settling in a 15 or 50 mL conical tube. They wererefed in serum-containing medium, and taken through cycles of clusterseparation, feeding, and reculturing every 2 or 3 days. After about 8days, there was considerably increased expression of cardiomyocytemarkers cTnI and MHC at the mRNA level (FIG. 7), and a high proportionof spontaneously contracting clusters (FIG. 8).

The Cardiac Body™ technique can be used to expand and/or enrich thecardiomyocytes in the cell population at any time in the differentiationprocess. As exemplified below, the technique can be used after aprevious enrichment step by density separation. Implementation of thetechnique has benefits that were not anticipated before the making ofthis invention. In particular, the expression of myosin heavy chaindetected by real-time PCR increases 10- to 100-fold when the cells arecultured for a 7 day period. A large proportion of the clusters in thecomposition exhibit spontaneous contractile activity: usually over 50%,and potentially between about 80% and 100% when processed in the mannerdescribed.

Characterization of Cardiomyocyte Lineage Cells

The cells obtained according to the techniques of this invention can becharacterized according to a number of phenotypic criteria.Cardiomyocytes and precursor cells derived from pPS cell lines oftenhave morphological characteristics of cardiomyocytes from other sources.They can be spindle, round, triangular or multi-angular shaped, and theymay show striations characteristic of sarcomeric structures detectableby immunostaining (FIG. 1). They may form flattened sheets of cells, oraggregates that stay attached to the substrate or float in suspension,showing typical sarcomeres and atrial granules when examined by electronmicroscopy.

pPS derived cardiomyocytes and their precursors typically have at leastone of the following cardiomyocyte specific markers:

-   -   Cardiac troponin I (cTnI), a subunit of troponin complex that        provides a calcium-sensitive molecular switch for the regulation        of striated muscle contraction    -   Cardiac troponin T (cTnT)    -   Nkx2.5, a cardiac transcription factor expressed in cardiac        mesoderm during early mouse embryonic development, which        persists in the developing heart        The cells will also typically express at least one (and often at        least 3, 5, or more) of the following markers:    -   Atrial natriuretic factor (ANF), a hormone expressed in        developing heart and fetal cardiomyocytes but down-regulated in        adults. It is considered a good marker for cardiomyocytes        because it is expressed in a highly specific manner in cardiac        cells but not skeletal myocytes.    -   myosin heavy chain (MHC), particularly the β chain which is        cardiac specific    -   Titin, tropomyosin, α-sarcomeric actinin, and desmin    -   GATA-4, a transcription factor that is highly expressed in        cardiac mesoderm and persists in the developing heart. It        regulates many cardiac genes and plays a role in cardiogenesis    -   MEF-2A, MEF-2B, MEF-2C, MEF-2D; transcription factors that are        expressed in cardiac mesoderm and persist in developing heart    -   N-cadherin, which mediates adhesion among cardiac cells    -   Connexin 43, which forms the gap junction between        cardiomyocytes.

β1-adrenoceptor (β1-AR)

-   -   creatine kinase MB (CK-MB) and myoglobin, which are elevated in        serum following myocardial infarction    -   α-cardiac actin, early growth response-I, and cyclin D2.

Tissue-specific markers can be detected using any suitable immunologicaltechnique—such as flow immunocytometry or affinity adsorption forcell-surface markers, immunocytochemistry (for example, of fixed cellsor tissue sections) for intracellular or cell-surface markers, Westernblot analysis of cellular extracts, and enzyme-linked immunoassay, forcellular extracts or products secreted into the medium. Antibodies thatdistinguish cardiac markers like cTnI and cTnT from other isoforms areavailable commercially from suppliers like Sigma and SpectralDiagnostics. Expression of an antigen by a cell is said to beantibody-detectable if a significantly detectable amount of antibodywill bind to the antigen in a standard immunocytochemistry or flowcytometry assay, optionally after fixation of the cells, and optionallyusing a labeled secondary antibody.

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods using publicly available sequence data (GenBank).Expression of tissue-specific markers as detected at the protein or mRNAlevel is considered positive if the level is at least 2-fold, andpreferably more than 10- or 50-fold above that of a control cell, suchas an undifferentiated pPS cell or other unrelated cell type.

Once markers have been identified on the surface of cells of the desiredphenotype, they can be used for immunoselection to further enrich thepopulation by techniques such as immunopanning or antibody-mediatedfluorescence-activated cell sorting.

Under appropriate circumstances, pPS-derived cardiomyocytes often showspontaneous periodic contractile activity. This means that when they arecultured in a suitable tissue culture environment with an appropriateCa⁺⁺ concentration and electrolyte balance, the cells can be observed tocontract across one axis of the cell, and then release from contraction,without having to add any additional components to the culture medium.The contractions are periodic, which means that they repeat on a regularor irregular basis, at a frequency between ˜6 and 200 contractions perminute, and often between ˜20 and ˜90 contractions per minute in normalbuffer (FIG. 2). Individual cells may show spontaneous periodiccontractile activity on their own, or they may show spontaneous periodiccontractile activity in concert with neighboring cells in a tissue, cellaggregate, or cultured cell mass.

The contractile activity of the cells can be characterized according tothe influence of culture conditions on the nature and frequency ofcontractions. Compounds that reduce available Ca⁺⁺ concentration orotherwise interfere with transmembrane transport of Ca⁺⁺ often affectcontractile activity. For example, the L-type calcium channel blockerdiltiazem inhibits contractile activity in a dose-dependent manner (FIG.2). On the other hand, adrenoceptor agonists like isoprenaline andphenylephrine have a positive chronotropic effect. Furthercharacterization of functional properties of the cell can involvecharacterizing channels for Na⁺, K⁺, and Ca⁺⁺. Electrophysiology can bestudied by patch clamp analysis for cardiomyocyte like actionpotentials. See Igelmund et al., Pflugers Arch. 437:669, 1999; Wobus etal., Ann. N.Y. Acad. Sci. 27:752, 1995; and Doevendans et al., J. Mol.Cell Cardiol. 32:839, 2000.

Functional attributes provide a manner of characterizing cells and theirprecursors in vitro, but may not be necessary for some of the usesreferred to in this disclosure. For example, a mixed cell populationenriched for cells bearing some of the markers listed above, but not allof the functional or electrophysiology properties, can be ofconsiderable therapeutic benefit if they are capable of grafting toimpaired cardiac tissue, and acquiring in vivo the functional propertiesneeded to supplement cardiac function.

Where derived from an established line of pPS cells, the cellpopulations and isolated cells of this invention can be characterized ashaving the same genome as the line from which they are derived. Thismeans that the chromosomal DNA will be over 90% identical between thepPS cells and the cardiac cells, which can be inferred if the cardiaccells are obtained from the undifferentiated line through the course ofnormal mitotic division. The characteristic that cardiomyocyte lineagecells are derived from the parent cell population is important inseveral respects. In particular, the undifferentiated cell populationcan be used for producing additional cells with a shared genome—either afurther batch of cardiac cells, or another cell type that may be usefulin therapy—such as a population that can pre-tolerize the patient to thehistocompatibility type of the cardiac allograft (US 2002/0086005 A1; WO03/050251).

Genetic Alteration of Differentiated Cells

The cells of this invention can be made to contain one or more geneticalterations by genetic engineering of the cells either before or afterdifferentiation (US 2002/0168766 A1). A cell is said to be “geneticallyaltered” when a polynucleotide has been transferred into the cell by anysuitable means of artificial manipulation, or where the cell is aprogeny of the originally altered cell that has inherited thepolynucleotide. For example, the cells can be processed to increasetheir replication potential by genetically altering the cells to expresstelomerase reverse transcriptase, either before or after they progressto restricted developmental lineage cells or terminally differentiatedcells (US 2003/0022367 A1).

The cells of this invention can also be genetically altered in order toenhance their ability to be involved in tissue regeneration, or todeliver a therapeutic gene to a site of administration. A vector isdesigned using the known encoding sequence for the desired gene,operatively linked to a promoter that is either pan-specific orspecifically active in the differentiated cell type. Of particularinterest are cells that are genetically altered to express one or moregrowth factors of various types such as FGF, cardiotropic factors suchas atrial natriuretic factor, cripto, and cardiac transcriptionregulation factors, such as GATA-4, Nkx2.5, and MEF2-C. Production ofthese factors at the site of administration may facilitate adoption ofthe functional phenotype, enhance the beneficial effect of theadministered cell, or increase proliferation or activity of host cellsneighboring the treatment site.

Use of Cardiomyocytes and their Precursors

This invention provides a method to produce large numbers of cells ofthe cardiomyocyte lineage. These cell populations can be used for anumber of important research, development, and commercial purposes.

Drug Screening

Cardiomyocytes of this invention can be used commercially to screen forfactors (such as solvents, small molecule drugs, peptides,oligonucleotides) or environmental conditions (such as cultureconditions or manipulation) that affect the characteristics of suchcells and their various progeny.

In some applications, pPS cells (undifferentiated or differentiated) areused to screen factors that promote maturation into later-stagecardiomyocyte precursors, or terminally differentiated cells, or topromote proliferation and maintenance of such cells in long-termculture. For example, candidate maturation factors or growth factors aretested by adding them to cells in different wells, and then determiningany phenotypic change that results, according to desirable criteria forfurther culture and use of the cells.

Other screening applications of this invention relate to the testing ofpharmaceutical compounds for their effect on cardiac muscle tissuemaintenance or repair. Screening may be done either because the compoundis designed to have a pharmacological effect on the cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on cells of this tissue type. The screening can be conductedusing any of the precursor cells or terminally differentiated cells ofthe invention.

The reader is referred generally to the standard textbook In vitroMethods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat.No. 5,030,015. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the differentiated cells of thisinvention with the candidate compound, either alone or in combinationwith other drugs. The investigator determines any change in themorphology, marker phenotype, or functional activity of the cells thatis attributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlates the effect of thecompound with the observed change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in In vitro Methods in PharmaceuticalResearch, Academic Press, 1997) for further elaboration.

Effect of cell function can be assessed using any standard assay toobserve phenotype or activity of cardiomyocytes, such as markerexpression, receptor binding, contractile activity, orelectrophysiology—either in cell culture or in vivo. Pharmaceuticalcandidates can also be tested for their effect on contractileactivity—such as whether they increase or decrease the extent orfrequency of contraction. Where an effect is observed, the concentrationof the compound can be titrated to determine the median effective dose(ED₅₀).

Animal Testing

This invention also provides for the use of cardiomyocytes and theirprecursors to enhance tissue maintenance or repair of cardiac muscle forany perceived need, such as an inborn error in metabolic function, theeffect of a disease condition, or the result of significant trauma.

To determine the suitability of cell compositions for therapeuticadministration, the cells can first be tested in a suitable animalmodel. At one level, cells are assessed for their ability to survive andmaintain their phenotype in vivo. Cell compositions are administered toimmunodeficient animals (such as nude mice, or animals renderedimmunodeficient chemically or by irradiation). Tissues are harvestedafter a period of regrowth, and assessed as to whether pPS derived cellsare still present.

This can be performed by administering cells that express a detectablelabel (such as green fluorescent protein, or β-galactosidase); that havebeen prelabeled (for example, with BrdU or [³H]thymidine), or bysubsequent detection of a constitutive cell marker (for example, usinghuman-specific antibody). The presence and phenotype of the administeredcells can be assessed by immunohistochemistry or ELISA usinghuman-specific antibody, or by RT-PCR analysis using primers andhybridization conditions that cause amplification to be specific forhuman polynucleotides, according to published sequence data.

Suitability can also be determined by assessing the degree of cardiacrecuperation that ensues from treatment with a cell population ofpPS-derived cardiomyocytes. A number of animal models are available forsuch testing. For example, hearts can be cryoinjured by placing aprecooled aluminum rod in contact with the surface of the anterior leftventricle wall (Murry et al., J. Clin. Invest. 98:2209, 1996; Reineckeet al., Circulation 100:193, 1999; U.S. Pat. No. 6,099,832; Reinecke etal., Circ Res., Epub March 2004). In larger animals, cryoinjury can beeffected by placing a 30-50 mm copper disk probe cooled in liquid N₂ onthe anterior wall of the left ventricle for ˜20 min (Chiu et al., Ann.Thorac. Surg. 60:12, 1995). Infarction can be induced by ligating theleft main coronary artery (Li et al., J. Clin. Invest. 100:1991, 1997).Injured sites are treated with cell preparations of this invention, andthe heart tissue is examined by histology for the presence of the cellsin the damaged area. Cardiac function can be monitored by determiningsuch parameters as left ventricular end-diastolic pressure, developedpressure, rate of pressure rise, and rate of pressure decay.

Therapeutic Use in Humans

After adequate testing, differentiated cells of this invention can beused for tissue reconstitution or regeneration in a human patient orother subject in need of such treatment. The cells are administered in amanner that permits them to graft or migrate to the intended tissue siteand reconstitute or regenerate the functionally deficient area. Specialdevices are available that are adapted for administering cells capableof reconstituting cardiac function directly to the chambers of theheart, the pericardium, or the interior of the cardiac muscle at thedesired location.

Where desirable, the patient receiving an allograft of pPS derivedcardiomyocytes can be treated to reduce immune rejection of thetransplanted cells. Methods contemplated include the administration oftraditional immunosuppressive drugs like cyclosporin A (Dunn et al.,Drugs 61:1957, 2001), or inducing immunotolerance using a matchedpopulation of pPS derived cells (WO 02/44343; U.S. Pat. No. 6,280,718;WO 03/050251). Another approach is to adapt the cardiomyocyte cellpopulation to decrease the amount of uric acid produced by the cellsupon transplantation into a subject, for example, by treating them withallopurinol. Alternatively or in conjunction, the patient is prepared byadministering allopurinol, or an enzyme that metabolizes uric acid, suchas urate oxidase (PCT/US04/42917).

Patients suitable for receiving regenerative medicine according to thisinvention include those having acute and chronic heart conditions ofvarious kinds, such as coronary heart disease, cardiomyopathy,endocarditis, congenital cardiovascular defects, and congestive heartfailure. Efficacy of treatment can be monitored by clinically acceptedcriteria, such as reduction in area occupied by scar tissue orrevascularization of scar tissue, and in the frequency and severity ofangina; or an improvement in developed pressure, systolic pressure, enddiastolic pressure, Δpressure/Δtime, patient mobility, and quality oflife.

The cardiomyocytes of this invention can be supplied in the form of apharmaceutical composition, comprising an isotonic excipient preparedunder sufficiently sterile conditions for human administration. When thedifferentiation procedure has involved culturing the cells as CardiacBodies™, it may be desirable to disperse the cells using a protease orby gentle mechanical manipulation into a suspension of single cells orsmaller clusters. To reduce the risk of cell death upon engraftment, thecells may be treated by heat shock or cultured with ˜0.5 U/mLerythropoietin ˜24 hours before administration.

For general principles in medicinal formulation, the reader is referredto Cell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996; and Hematopoetic Stem Cell Therapy, E. D. Ball, J. Lister &P. Law, Churchill Livingstone, 2000. Choice of the cellular excipientand any accompanying elements of the composition will be adapted inaccordance with the route and device used for administration. Thecomposition may also comprise or be accompanied with one or more otheringredients that facilitate the engraftment or functional mobilizationof the cardiomyocytes. Suitable ingredients include matrix proteins thatsupport or promote adhesion of the cardiomyocytes, or complementary celltypes, especially endothelial cells.

This invention also includes a reagent system, comprising a set orcombination of cells that exist at any time during manufacture,distribution, or use. The cell sets comprise any combination of two ormore cell populations described in this disclosure, exemplified but notlimited to a type of differentiated pPS-derived cell (cardiomyocytes,cardiomyocyte precursors, Cardiac Bodies™, and so on), in combinationwith undifferentiated pPS cells or other differentiated cell types,often sharing the same genome. Each cell type in the set may be packagedtogether, or in separate containers in the same facility, or atdifferent locations, at the same or different times, under control ofthe same entity or different entities sharing a business relationship.

Pharmaceutical compositions of this invention may optionally be packagedin a suitable container with written instructions for a desired purpose,such as the reconstitution of cardiomyocyte cell function to improve adisease condition or abnormality of the cardiac muscle.

The following examples are provided as further non-limitingillustrations of particular embodiments of the invention.

EXAMPLES Example 1 Differentiation of hES Cells to Cardiomyocytes

hES cell lines, H1, H7, H9, and H9.2 (a cloned line derived from H9)were initially established on feeder cells and later maintained underfeeder-free conditions, as described in WO 01/51616. Differentiation wasinitiated by culturing hES cells in suspension to form embryoid bodies.After four days in suspension culture, the EBs were transferred togelatin-coated plates or chamber slides. Beating cardiomyocytes wereisolated from EB outgrowth mechanically at differentiation day 15-29,harvested, and washed. All hES cell lines tested had the potential togenerate beating cardiomyocytes, even after being maintained for over 50passages (˜260 population doublings), although some lines (e.g., H7)generated more than others.

FIG. 1 shows immunocytochemistry of cells suspended using Collagenase Band replated, then stained for expression of sarcomeric myosin heavychain (MHC), titin, tropomyosin, α-sarcomeric actinin, desmin, cTnI andcardiac troponin T (cTnT). Single cells and clusters stained positivefor all these markers. The stained single cardiomyocytes were spindle,round and tri- or multi-angular shaped. The striations characteristic ofthe sarcomeric structures is also seen, consistent with the contractileapparatus necessary for muscle function.

Cardiomyocytes were further enriched by density separation on adiscontinuous gradient of Percoll™ (a density separation mediumcomprising colloidal PVP-coated silica). Cardiomyocytes were generatedas before, harvested after 15 days plated on gelatin, and resuspended inthe differentiation medium. After settling for 5 min, the cellsuspension was loaded onto a layer of 40.5% Percoll™ (Pharmacia) (˜1.05g/mL) overtop of a layer of 58.5% Percoll™ (˜1.075 g/mL). The cells werethen centrifuged at 1500 g for 30 min. After centrifugation, cells atthe interface of the two layers were harvested, washed, resuspended indifferentiation medium, and seeded into chamber slides. The harvestedcells showed 26.8±4.1% staining for sarcomeric myosin heavy chain (MHC),which is at least ˜20-fold higher than the starting cell population.

The function of hES-derived cardiomyocytes was tested by determiningwhether the cardiomyocytes respond appropriately to the chronotropiceffects of cardioactive drugs.

FIG. 2 (Panel A) shows that the beating rate was inhibited by the L-typecalcium blocker diltazem in a concentration-dependent manner. When cellswere treated with 10⁻⁵ M diltiazem, 100% of the beating areas stoppedcontraction. This observation shows that the hES-derived cardiomyocyteshave functional L-type calcium channels. Panels B and C show that thereare positive chronotropic effects induced by isoprenaline (aβ-adrenoceptor agonist) and phenylephrine (an α-adrenoceptor agonist).Panels D and E show that the phosphodiesterase inhibitor IBMX and theβ2-adrenoceptor agonist clenbuterol have a similar effect. Thus, the hEScell derived cells respond to cardioactive drugs in a manner appropriatefor cells of the cardiomyocyte lineage.

Example 2 Factors that Promote Cardiomyocyte Differentiation

Embryoid bodies from the H1 or H9 line were treated at differentiationday 1-4, 4-6 or 6-8 with 5-aza-deoxy-cytidine, a cytosine analog thataffects DNA methylation. Cells were harvested at day 15, and analyzedfor cardiac MHC by real-time RT-PCR. One to 10 μM of5-aza-deoxy-cytidine at day 6-8 significantly increased the expressionof cardiac α-MHC, correlating with an increased proportion of beatingareas in the culture.

Other reagents examined for an ability to induce cardiomyocytedifferentiation included dimethyl sulfoxide (DMSO) and all-transretinoic acid (RA). Embryoid bodies treated with 0.5% DMSO from days 0-4produced fewer beating areas than non-treated cultures. Beating cellswere absent from cultures treated with 0.8% or 1% DMSO, and 1.5% DMSOwas actually toxic to the cells. DMSO treatment also caused significantreduction in α-MHC expression, compared with untreated cultures.

Retinoic acid was applied to differentiating hES cultures at dosesbetween 10⁻⁹ and 10⁻⁵ M. At day 0-4, the RA was toxic to the cells,while at days 4-8, 8-15, or 4-15, there was no increase in beating cellscompared with untreated cultures. Thus, 5-aza-deoxy-cytidine increasedthe proportion of cardiomyocyte cells in the population. In contrast,DMSO and retinoic acid inhibit cardiomyocyte differentiation, eventhough these compounds generate cardiomyocytes from mouse embryoniccarcinoma or embryonic stem cells (Wobus et al., J. Mol. Cell Cardiol.29:1525, 1997; McBurney et al., Nature 299:165, 1982).

Example 3 Effective Combinations of Cardiomyocyte Differentiation Agents

This example is an investigation of combined effects of added growthfactors to influence cardiomyocyte differentiation of human ES cells.

The rationale was as follows. Group I factors were selected as beingable to supply functions of the hypoblast during initial commitment.Group II factors were selected as able to supply functions of endodermduring subsequent development in combination with Group I factors. GroupIII factors were selected as survival factors for cardiomyocytes inextended culture. A typical working concentration was defined as“medium” level, with 4-fold lower and 4-fold higher levels defined as“low” and “high” levels, The concentrations are shown below:

TABLE 2 Exemplary Cardiotropic Factors Low Medium High Growth Factorconcentration concentration concentration Group I Activin A 6.25 ng/mL25 ng/mL 100 ng/mL TGF β1 2.5 ng/mL 10 ng/mL 40 ng/mL IGF II 6.25 nM 25nM 100 nM Group II BMP 4 1.25 ng/mL 5 ng/mL 20 ng/mL FGF 4 12.5 ng/mL 50ng/mL 200 ng/mL Insulin 6.25 ng/mL 25 ng/mL 100 ng/mL bFGF 12.5 ng/mL 50ng/mL 200 ng/mL PDGF-BB 12.5 ng/mL 50 ng/mL 200 ng/mL5-aza-deoxy-cytidine 10 μM 10 μM 10 μM Group III IGF I 6.25 nM 25 nM 100nM IGF II 6.25 nM 25 nM 100 nM LIF 5 ng/mL 20 ng/mL 80 ng/mL EGF 6.25ng/mL 25 ng/mL 100 ng/mL PDGF-BB 0.9 ng/mL 3.6 ng/mL 14.4 ng/mL bFGF 2.5ng/mL 10 ng/mL 40 ng/mL Insulin 6.25 nM 25 nM 100 nM

FIG. 3 (Upper Panel) shows the scheme for use of these factors. H1 cellsat passage 48 were used to generate embryoid bodies by collagenasetreatment followed by mechanically dislodging the cells from the dish byscraping with a 5 mL pipet. The contents of one 10 cm² well of cells wastransferred to a single 10 cm² well of a low adherence plate andcultured in 4 ml of DMEM plus 20% FBS in the presence or absence ofadditional factors for 4 days. At the end of day 4, each suspension ofembryoid bodies was divided into 2 aliquots plated in 2 wells of agelatin-coated adherent 6 well tissue culture plate (10 cm²/well). Theadherent embryoid bodies and their outgrowths were cultured in 4 mL ofDMEM plus 20% FBS in the presence or absence of additional factors for11 days, after which the number of beating regions in each well wasobserved by light microscopy, and RNA was harvested from each well forsubsequent quantitative PCR analysis.

Group I factors were added on day 0, (the day on which undifferentiatedcells were transferred to suspension culture to generate embryoidbodies) and were present continuously until day 8 (4 days after theembryoid bodies were plated in gelatin-coated wells). Group II factorswere added on day 4 (at the time of plating) and were presentcontinuously until day 8. Group III factors were added on day 8 and werepresent continuously until the end of the experiment (day 15). A subsetof cultures was exposed to 5-aza-deoxy-cytidine for 48 hrs (day 6-8).Cultures were re-fed with fresh media plus or minus factors on days 6,8, 11, and 13.

It was observed that while no beating regions were observed in thecontrol cultures (those maintained in the absence of supplementaryfactors/5-aza-deoxy-cytidine) or those maintained in the presence of thegrowth factors in the absence of 5-aza-deoxy-cytidine, beating areaswere observed in all wells receiving the combination of growth factorsplus 5-aza-deoxy-cytidine.

FIG. 3 (Lower Panel) shows quantitative PCR analysis (Taqman™) forexpression of the cardiac gene a myosin heavy chain (αMHC), relative tothe level in normal heart RNA. The level of expression was significantlyhigher in cells exposed to growth factors (GF) plus5-aza-deoxy-cytidine. The lowest concentrations tested were sufficientto achieve higher αMHC expression (30-fold higher than the levels seenin control.

These results were elaborated in a subsequent experiment. H1 cells(passage 38) were cultured as before, except that: a) only the lowestconcentrations of factors used in the previous experiment were employed;and b) in one set of samples, the Group III treatment was omitted. Thelevel of marker expression was then determined in real-time PCR assayrelative to undifferentiated cells.

FIG. 4 shows that omission of Group III from the protocol led to afurther 3-fold increase in the amount of αMHC mRNA expression. Increasesin the expression of the early cardiomyocyte-associated gene GATA-4 werealso detected. In contrast, the endoderm-associated gene HNF3b is notspecifically induced under these conditions. The effect on α-MHC andGATA-4 was selective, in comparison with the endoderm-associated geneHNF3b, which increased using any growth factor combination, but not with5-aza-deoxy-cytidine.

These results demonstrate that factors within Groups I and II enhancethe proportion of cells bearing characteristic features ofcardiomyocytes.

Example 4 Four-Phase Centrifugation Separation Method

Cardiomyocytes were generated from hES cells of the H7 line by formingembryold bodies for 4 days, and then proliferating on gelatin-coatedplates for 17 days (5-aza-deoxy-cytidine and growth factors were notused). The cells were then dissociated using collagenase B, resuspendedin differentiation medium. The cell suspension was then layered onto adiscontinuous gradient of Percoll™, and centrifuged at 1500 g for 30min. Four fractions were collected: I. The upper interface; II. The40.5% layer; III. The lower interface; IV. The 58.5% layer. The cellswere washed and resuspended in differentiation medium. Cells forimmunostaining were seeded into chamber slides at 10⁴ cells per well,cultured for 2 or 7, and then fixed and stained.

Results are shown in Table 3. Percentage of MHC positive cells wasdetermined by counting cells in 30 images from triplicate wells for eachfraction and presented as mean±standard deviation of cells from 3 wells.

TABLE 3 Percoll ™ Separation % staining for MHC Fraction Cell CountBeating Cells Day 2 Day 7 Before + 17 ± 4.4% 15 ± 4%  separation I 9.0 ×10⁶ ± 2.6 ± 0.5% 2.5 ± 3.0% II 1.6 × 10⁶ + 4.5 ± 1.5% 2.4 ± 0.9% III 4.0× 10⁶ ++ 35.7 ± 2.7%  28.3 ± 9.4%  IV 1.3 × 10⁶ +++ 69. ± 5.0% 52.2 ±14.5%Beating cells were observed in all fractions, but Fractions III and IVcontained the highest percentage.

Phenotype of the cells as determined by indirect immunocytochemistry isshown in Table 4.

TABLE 4 Characteristics of Separated Cell Populations EpitopeCardiomyocyte lineage Non-cardiac cells cTn1 ++ − cardiac-specific α/βMHC ++ − cardiac β MHC ++ − sarcomeric MHC ++ − N-cadherin ++ ± smoothmuscle actin ++ subset myogenin − − α-fetoprotein − − β-tubulin III − −Ki67 subset subset BrdU subset subset SSEA-4 − − Tra-1-81 − −Cardiomyocyte populations separated by density gradient centrifugationcould be distinguished by staining for cTnI and MHC. Absence of stainingfor myogenin, α-fetoprotein, or β-tubulin III showed the absence ofskeletal muscle, endoderm cell types, and neurons. Lack of staining forSSEA-4 and Tra-1-81 confirms the absence of undifferentiated hES cells.

α-Smooth muscle actin (SMA) is reportedly present in embryonic and fetalcardiomyocytes, but not adult cardiomyocytes (Leor et al., Circulation97:I1332, 1996; Etzion et al., Mol. Cell Cardiol. 33:1321, 2001).Virtually all cTnI-positive cells and a subset of cTnI negative cellsobtained in the cardiomyocyte differentiation protocol were positive forSMA, suggesting that they may be at an early stage and capable ofproliferation.

Cells in fraction III and IV were replated, cultured for an additional 2days. 43±4% of the MHC positive cells expressed BrdU, indicating thatthey were in the S phase of the cell cycle. In other experiments, asubset of cTnI-positive cells were found to express Ki-67. These resultsshow that about 20% or 40% of the cardiomyocytes in the population wereundergoing active proliferation.

Example 5 Direct Differentiation Protocol

In this example, hES cells of the H7 line were differentiated intocardiomyocyte lineage cells by plating onto a substrate and culturing ina serum-free medium containing differentiation factors.

Tissue culture surfaces were prepared by coating overnight with 0.5%gelatin, then incubating for 2 to 4 h with medium containing 20% FBS,which was removed prior to plating of the hES cells. Alternatively, theplastic was coated with human fibronectin (20 μg/mL) or growthfactor-reduced Matrigel® with no subsequent incubation withserum-containing medium.

In an exemplary trial, TGF-β related factors were tested for theirability to induce expression of genes characteristic of mesoderm orearly stage cardiomyocytes. Undifferentiated hES cells from the H7 linewere seeded into 24 well gelatin coated plates. After one week of growthin mEF conditioned medium as undifferentiated cells, the medium waschanged to RPMI+B27 supplement, with or without 50 ng/mL Activin A, 50ng/mL BMP-4, or both factors together. After four days, the growthfactors were removed by medium exchange, and the cells were thencultured for an additional 14 days in RPMI+B27 alone. For comparison,hES cells were also differentiated by the embryoid body protocol asalready described (culturing four days suspended in medium containing20% FBS, then on 0.5% gelatin-coated surfaces, and harvesting at day12-20 of culture).

Expression of α-myosin heavy chain in the differentiated cells wasdetermined by real-time PCR analysis using gene-specific primers. Datawere normalized by multiplex reactions with the 18s rRNA-specific assayfrom Applied Biosystems.

FIG. 5(A) shows the results. The combination of Activin A and BMP-4 inthe direct differentiation method on gelatin coated plates producedcells expressing considerably higher levels of MHC, compared with cellsgenerated from embryoid bodies in serum-containing medium. Numerousspontaneously beating areas were evident, forming spheres that latertook on a more flattened appearance and began to beat 7 days afterwithdrawing Activin A and BMP-4. No such beating areas were evident inwells cultured without Activin A and BMP-4.

In a separate trial, cells were differentiated in the same fashion onmultiwell chamber slides. Multiple spontaneously beating areas wereevident in wells cultured with using Activin A and BMP-4, showingevidence of organized, functional sarcomeres, whereas no beating areaswere present in wells cultured in the absence of the factor combination.The slides were fixed with 2% paraformaldehyde, permeabilized withethanol, and then stained for marker expression.

FIG. 5(B) shows the results. The cells demonstrated both strongnuclear-localized expression of the cardiac transcription factor Nkx2.5,and cytoplasmic expression of α-sarcomeric actinin. Clusters of cellswere observed that stained for both markers. Separate positivelystaining cells had organized striated patterns of α-actinin, consistentwith functional sarcomeres. Similar results were obtained using the H1line of hES cells.

In another experiment, cardiomyocytes were generated from the H7 line ofhES cells previously expanded in the undifferentiated form in fresh(non-conditioned) medium. The defined medium was made from commerciallyobtained XVIVO-10™ (BioWhittaker), as described in US 2005/0037492 A1,supplemented with 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 0.1 mMnon-essential amino acids (invitrogen), 80 ng/mL bFGF and 0.5 ng/mLTGFβ1.

The first step was to plate hES cells onto matrix suitable forcardiomyocyte generation. Confluent H7 hES cells maintained in thedefined medium were incubated with collagenase IV (200 U/mL) at 37° C.for 5 min, washed with PBS, harvested as small clusters, and plated ontogelatin coated chamber slides or 24 well plates (coated with 0.5%gelatin overnight, then incubated with FBS containing medium overnight).The cells were cultured on the new matrix for two days in mediumcontaining 20% FBS, 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 0.1mM non-essential amino acids followed by XVIVO-10™ medium, followed byfour days in XVIVO-10™ medium containing 8 or 80 mg/mL bFGF and 0.5ng/mL TGFβ1 for 6 days; or for six days in the supplemented XVIVO-10™medium.

To differentiate into cardiomyocytes, the plated cells were nextcultured in RPMI medium supplemented with B27, 50 ng/mL BMP-4 and 50ng/mL Activin A. The medium was replaced after four days with RPMImedium supplemented with B27, but lacking the growth factors.

Cells at differentiation day 18 were harvested for immunocytochemicalanalysis, They were fixed in PBS containing 2% paraformaldehyde,permeabilized with ethanol, blocked with 10% normal goat serum, and thenincubated with antibody against Nkx2.5 (Santa Cruz Biotech) orα-sarcomeric actinin (Sigma), followed by labeled secondary antibody.

FIG. 6 shows the results. Cardiac lineage cells were identified byexpression of both Nkx2.5 in the nuclei and α-sarcomeric actinin in thecytoplasm. Some of the cells present expressed Nkx2.5 in the nuclei butwere negative for α-actinin expression, which is believed to representcardiomyocyte precursors cells. Both the double positive cells and theNkx2.5 positive/α-actinin negative cells were present as cell clumps ofvarious sizes.

The direct differentiation method produces large numbers of beatingcells arise that are suitable for both in vitro studies andtransplantation. The surprising efficiency and ease of cardiomyocytegeneration in this system implies a high proportion of cardiomyocyteprogenitors, which is valuable for certain aspects of commercial scalecardiomyocyte production, and the use of these cells for drug screeningand therapy.

Example 6 Enrichment of Contracting Cells by Making Cardiac Bodies™

This example illustrates the subsequent culturing of cardiomyocyteclusters as Cardiac Bodies™ to enrich for cells having characteristicsdesirable for therapeutic use and other purposes.

Three 225 cm² flasks of undifferentiated hES cells of the H7 line wereused to generate embryoid bodies as already described. The EBs from eachflask were resuspended in 75 mL of medium and transferred to three lowadhesion six well plates (4 mL cell suspension per well), yielding nineplates of EBs in suspension in total. The EBs were re-fed after one dayin suspension by transferring the newly formed EBs to 50 mL conicaltubes (one plate per tube), letting the EBs settle at room temperaturewithout agitation for 10 to 20 min, then removing the medium andreplacing with fresh medium (25 mL per tube).

The EBs were returned to their original low attachment plates andmaintained in suspension in 20% FBS containing medium for 3 additionaldays, then transferred to a total of three gelatin-coated 225 cm² tissueculture flasks. Two days after transfer to the gelatin coated flasks,the medium was removed and each flask was re-fed with 75 mL 20% FBScontaining medium. Similar re-feedings occurred on day 8, 11, 13, 15,and 18. On day 20, the differentiated cultures were dissociated withBlendzyme™ and fractionated on discontinuous Percoll™ gradients asbefore. Fraction IV (the highest density fraction) was recovered andcounted, yielding ˜3.7×10⁶ single cells and small clusters.

The Fraction IV cells were resuspended in ˜6.5 mL of 20% FBS containingmedium, transferred to a 15 mL conical tube, and allowed to settle atroom temperature without agitation for 10 min. The medium (containing2.8×10⁶ cells, which is most of the single cells) was removed andreplaced with fresh medium. The cell suspension was transferred to asingle low attachment six well plate (˜4 mL of cell suspension perwell). The CBs were re-fed in a similar manner (transfer to 50 mL tube,settling for 10 min, medium removal and replacement) every 48 h.

FIG. 7 shows the expression of the sarcomeric genes αMHC and cardiactroponin I as measured by real-time PCR (Taqman™). Relative to theexpression after 20 days of culture on gelatin, separating the cells byPercoll™ increased expression by 2-5 fold in Fraction IV cells. Removingthe single cells and collecting clusters increased expression to 5-20fold. After 8 days of culturing the cells as Cardiac Bodies™, theexpression was 100- to 500-fold higher than the unseparated cells.

When CBs are replated onto gelatin or Matrigel® (isolated basementmembrane produced by Engelbreth-Holm-Swarm tumor cells and containingextracellular matrix components such as laminin), the clusters adhere,flatten, and produce large patches of spontaneously contracting cells.For use in animal testing, the Cardiac Bodies™ may be implanteddirectly, or dispersed into suspensions of single cells.

Example 7 Comparison of Culture Conditions

In this example, the cardiomyocyte differentiation culture was conductedfor different periods before Percoll™ separation and Cardiac Body™formation.

Seven 225 cm² flasks of undifferentiated hES cells were used to generateEBs, yielding 21 plates of EBs in suspension in total. As before, theEBs were cultured in 20% fetal bovine serum, plated onto gelatin on day4, and refed with fresh medium every 2 or 3 days thereafter. On day 12,four flasks of differentiated cells were separated by density gradientcentrifugation as before, and on day 20, the remaining 3 flasks wereprocessed. Clustered cells in each of the four Percoll™ fractions wereseparated into single cell suspensions. They were then grown insuspension culture to form Cardiac Bodies™, fed with fresh medium ondays 2, 5, and 6. On day 7, they were harvested and viewed under themicroscope.

FIG. 8(A) shows a field of Cardiac Bodies™ made from Fraction IV cells(bar≡300 μm). The clusters marked by the arrows were undergoingspontaneous contractions. FIG. 8(B) shows the quantitative data obtainedby counting the contracting clusters in each preparation. Fraction IVshowed the highest proportion of spontaneously contracting cells, andwas higher when the starting population had been differentiated for 20days. Using a similar protocol, suspensions have been obtained in whichmost of the clusters were beating.

It was found that the percentage of cardiac cells in Cardiac Bodies™ canbe increased as follows: after the Percoll™ gradient separation andremoval of single cells from the fraction IV clusters, the clusters aredissociated to a single cell suspension by trypsinizing and resuspendingin culture medium (20% FBS-containing medium or preferably a serum-freemedium containing a serum substitute, or CCT). The suspension istransferred to low-adhesion 6 well plates (4 mL/well) and cultured withre-feeding every 2-3 days. The resultant “secondary” Cardiac Bodies™that form show a higher percentage of cardiomyocytes (45.9%, determinedby flow cytometry for cTNT-positive cells) compared with the clustersthat formed initially (14.1%).

In subsequent experiments, Cardiac Bodies™ were analyzed for phenotypicmarkers, with the following results.

TABLE 4 Characteristics of Cardiac Bodies ™ Epitope Cardiomyocytelineage Non-cardiac cells cTnl ++ − cTnT ++ − α-actinin ++ − sarcomericMHC ++ − CD56 ++ subset Pan-cytokeratins − subset

The techniques of direct differentiation and cardiac bodyT™ formationcan be combined to optimize purity and yield of cardiomyocyte lineagecells. Exemplary is the following procedure.

Undifferentiated hES cells are plated onto tissue culture plates orflasks that have been pretreated with Matrigel®, human fibronectin, or0.5% gelatin, preincubated with FBS. Cells are expanded either with mEFconditioned medium, or with XVIVO-10™ medium supplemented with 100 ng/mLbFGF and 0.5 ng/mL TGFβ1). After 1 week, the medium is replaced withRPMI plus a supplement like B27, and 50 ng/mL each of Activin A andBMP-4. Four days later, the medium is replaced with RPMI plus supplementwithout the Activin or BMP. Cultures are fed every 2 or 3 days with thesame medium until the cell harvest (typically ˜14 days after the removalof activin and BMP-4).

Cells are harvested and purified by Blendzyme™ digestion andcentrifugation through a discontinuous Percoll™ gradient. Fraction IVcells are resuspended at a concentration corresponding to approximately1-5 million cells/mL and transferred to a conical tube. The cellsuspension is incubated at room temperature for 10 min withoutagitation. The floating cells are removed by gentle aspiration and theremaining clusters are washed once with PBS and then dissociated tosingle cells with 0.025% trypsin/EDTA. The cells are resuspended in 20%FBS-containing medium at approximately 4 mL per 5 million startingFraction IV cells. The cell suspension is transferred to low adhesiontissue culture plates and re-fed every 2 to 4 days by gentlycentrifuging or letting the Cardiac Bodies™ settle at room temp prior toreplacement of the medium. The Cardiac Bodies™ can be used as anenriched source of hES-derived cardiomyocytes after 1-2 weeks ofsuspension culture, or subject to further rounds of Cardiac Body™formation before harvesting.

Example 8 Transplantation of Cardiac Bodies™ into the Intact Myocardium

To assess the ability of cardiac bodies to survive in vivo, H7-derivedcells were implanted into uninjured hearts of adult nude rats. H7 hEScells were used to generate embryoid bodies in 20% FBS-containingmedium; the embryoid bodies were cultured in suspension for 4 days, andthen allowed to adhere to gelatin-coated flasks, where they werecultured for 2 additional weeks. Cardiac Bodies™ were prepared asdescribed above, and maintained in suspension in 20% FBS-containingmedium for 1 week with re-feeding every 2-3 days.

The implantation experiments were done by Drs. Charles Murry and MichaelLaflamme at the University of Washington in Seattle, under a SponsoredResearch Agreement with Geron Corp. Twenty-four hours prior toimplantation, the Cardiac Bodies™ were subjected to 30 minute heat shockat 43° to increase survival. On the day of implantation, Cardiac Bodies™were injected into the left ventricular myocardium of uninjured nuderats. After 1 week, the rats were sacrificed, the hearts were fixed,sectioned, and examined for the presence of human cells in themyocardium.

FIG. 9 shows the results. Human cells were identified in two out ofthree of the rats on the basis of in situ hybridization with ahuman-specific pancentromeric probe (middle panel). The human cells werefurther identified as cardiomyocytes by labeling with an antibodydirected against β-myosin heavy chain and specific for the humanortholog (lower panel).

These data demonstrate that the hES derived cardiomyocyte lineage cellsof this invention are suitable for transplantation into the myocardium,where they survive and integrate into the host tissue.

The Compositions and Procedures Provided in the Description can beEffectively Modified by Those Skilled in the Art without Departing fromthe Invention Embodied in the Claims that Follow

1. A method of differentiating a primate pluripotent stem (pPS) cellinto a cell expressing α-MHC comprising a) plating the undifferentiatedpPS cell without forming an embryoid body directly onto a solid surfacecomprising a substrate; b) contacting the pPS cell plated on the solidsurface with one of the following: 1) BMP; or 2) activin and BMP; c)culturing the contacted pPS cell of b), thereby directingdifferentiation of the pPS cell into a cell expressing α-MHC.
 2. Themethod of claim 1, wherein the pPS cell is cultured serum-free in stepsb) and c).
 3. The method of claim 1, wherein the pPS cell is contactedwith BMP.
 4. The method of claim 1, wherein the pPS cell is contactedwith activin and BMP.
 5. The method of claim 1, wherein the substrate isproduced by Engelbreth-Holm-Swarm tumor cells.
 6. The method of claim 1,wherein the substrate is gelatin.
 7. A method of differentiating aprimate pluripotent stem (pPS) cell into a cell expressing Nkx2.5comprising a) plating the undifferentiated pPS cell without forming anembryoid body directly onto a solid surface comprising a substrate; b)contacting the pPS cell with activin and BMP; c) culturing the contactedpPS cell of b), thereby directing differentiation of the pPS cell into acell expressing Nkx2.5.
 8. The method of claim 7, wherein the pPS cellis cultured serum-free in steps b) and c).
 9. The method of claim 7,wherein the substrate is produced by Engelbreth-Holm-Swarm tumor cells.10. The method of claim 7, wherein the substrate is gelatin.
 11. Amethod of differentiating a primate pluripotent stem (pPS) cell into acell expressing α-sarcomeric actinin comprising a) plating theundifferentiated pPS cell without forming an embryoid body directly ontoa solid surface comprising a substrate; b) contacting the pPS cell withactivin and BMP; c) culturing the contacted pPS cell of b), therebydirecting differentiation of the pPS cell into a cell expressingα-sarcomeric actinin.
 12. The method of claim 11, wherein the pPS cellis cultured serum-free in steps b) and c).
 13. The method of claim 11,wherein the substrate is produced by Engelbreth-Holm-Swarm tumor cells.14. The method of claim 11, wherein the substrate is gelatin.